Plant tubby-like proteins

ABSTRACT

An isolated polypeptide containing an amino acid sequence at least 70% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, and an isolated nucleic acid encoding the polypeptide are disclosed. Disclosed is an isolated nucleic acid that, under stringent conditions, hybridizes to a probe containing SEQ ID NO:20; or its complementary sequence. Also disclosed are (1) a transformed cell or a transgenic plant containing such a nucleic acid and (2) a transformed cell or a transgenic plant lacking the polypeptide encoded by the nucleic acid. Also within the scope of the invention are methods for making the transformed cells or transgenic plants.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 60/441,380, filed on Jan. 21, 2003, the contents of which areincorporated by reference in their entirety.

BACKGROUND

Various environmental factors, e.g., high salinity, pathogens, andchilling, cause stress and adverse effects on growth and productivity ofcrops. It is therefore desirable to produce transgenic crops that aretolerant to such factors. Genetic engineering can be used to modifyproteins that are involved in regulating responses of plants toenvironmental factors, thereby improving stress-tolerance.

TUBBY proteins, a group of membrane-bound transcription regulators, werefirst identified from obese mice via positional cloning (Kleyn et al.,1996, Cell 85: 281-290 and Noben-Trauth et al., 1996, Nature 380:534-538.). Mutations in the TUBBY genes lead to maturity-onset obesity,insulin resistance, retinal degeneration, and neurosensory hearing loss.TUBBY-like proteins (TLPs) were subsequently discovered in other mammalsand were found to be activated through G-proteins, which, in higherplants, are involved in the response to environmental factors andhormone regulation (Warpeha et al., 1991, Proc. Natl. Acad. Sci. 88:8925-8929, and Ueguchi-Tanaka et al., Proc. Natl. Acad. Sci. 97:11638-11643).

SUMMARY

This invention is based on the discovery of eleven ArabidopsisTUBBY-like proteins, designated as AtTLPs 1-11. These proteins regulatethe response of Arabidopsis to environmental factors. The full-lengthAtTLPs 1-11 polypeptides (designated as SEQ ID NOs: 1-11), and cDNAsencoding the polypeptides (designated as SEQ ID NOs: 12-22), are shownbelow:

AtTLP1: polypeptide:   1 MSFRSIVRDV RDSIGSLSRR SFDFKLSSLN KEGGKSRGSVQDSHEEQLVV (SEQ ID NO: 1)  51 TIQETPWANL PPELLRDVIK RLEESESVWPARRHVVACAS VCRSWRDMCK  101 EIVQSPELSG KITFPVSLKQ PGPRDATMQC FIKRDKSNLTYHLYLCLSPA  151 LLVENGKFLL SAKRIRRTTY TEYVISMHAD TISRSSNTYI GKIRSNFLGT 201 KFIIYDTQPA YNSNIARAVQ PVGLSRRFYS KRVSPKVPSG SYKIAQVSYE  251LNVLGTRGPR RMHCAMNSIP ASSLAEGGTV PGQPDIIVPR SILDESFRSI  301 TSSSSRKITYDYSNDFSSAR FSDILGPLSE DQEVVLEEGK ERNSPPLVLK  351 NKPPRWHEQL QCWCLNFRGRVTVASVKNFQ LIAANQPQPQ PQPQPQPQPL  401 TQPQPSGQTD GPDKIILQFG KVGKDMFTMDFRYPLSAFQA FAICLSSFDT  451 KLACE cDNA:   1 ATGTCGTTCC GTAGCATAGTTCGTGATGTG AGAGATAGTA TAGGAAGTCT (SEQ ID NO: 12)  51 ATCGAGGCGTAGTTTCGACT TTAAGTTAAG CAGCTTGAAC AAAGAAGGTG  101 GTAAATCCCG TGGTTCGGTTCAAGATTCTC ATGAGGAACA ACTTGTAGTA  151 ACGATTCAAG AAACACCGTG GGCGAATCTACCTCCAGAGT TATTACGTGA  201 TGTGATCAAA AGACTTGAAG AGAGTGAAAG TGTGTGGCCTGCTCGTAGAC  251 ATGTTGTTGC TTGTGCTTCT GTTTGCAGGT CATGGAGAGA TATGTGTAAA 301 GAGATTGTTC AAAGTCCGGA GCTCTCAGGC AAAATCACAT TTCCTGTTTC  351GTTGAAACAG CCTGGACCAA GAGATGCAAC AATGCAATGC TTTATCAAAA  401 GGGATAAATCTAACTTGACT TATCATTTAT ATCTTTGTCT CAGTCCTGCT  451 TTGTTGGTTG AGAATGGAAAGTTTCTTCTT TCTGCAAAAC GCATAAGAAG  501 AACTACATAC ACCGAGTACG TGATCTCTATGCACGCCGAC ACCATTTCGA  551 GATCAAGCAA TACCTACATT GGCAAAATCA GGTCTAATTTTCTGGGGACG  601 AAGTTTATAA TATACGATAC ACAACCAGCA TACAACAGCA ACATCGCTCG 651 AGCGGTCCAA CCGGTAGGTC TTAGCCGCAG ATTCTACTCA AAGAGAGTCT  701CTCCCAAAGT ACCTAGTGGG AGCTACAAAA TTGCGCAGGT TTCTTATGAG  751 CTAAACGTTCTTGGTACCCG TGGTCCGAGG AGAATGCATT GTGCGATGAA  801 CTCAATTCCC GCCTCTTCCCTTGCGGAAGG CGGAACTGTG CCTGGACAGC  851 CCGATATCAT TGTCCCGCGC TCTATTCTCGACGAATCGTT CCGCAGCATT  901 ACCTCTTCGT CATCGAGAAA AATCACTTAC GATTACTCGAATGATTTTAG  951 CAGTGCACGC TTTTCCGACA TTCTTGGCCC GTTAAGCGAA GACCAAGAAG1001 TGGTATTAGA AGAAGGGAAA GAGCGGAATT CGCCACCACT TGTGCTTAAG 1051AACAAGCCGC CGAGGTGGCA TGAACAGCTT CAGTGTTGGT GTTTAAACTT 1101 CAGGGGACGTGTAACAGTCG CATCAGTTAA GAACTTTCAG CTCATTGCAG 1151 CAAACCAACC ACAGCCTCAGCCTCAGCCTC AACCGCAACC TCAACCCCTA 1201 ACTCAGCCGC AACCGTCTGG TCAGACCGATGGTCCCGACA AGATCATATT 1251 GCAGTTTGGG AAAGTGGGAA AAGACATGTT CACGATGGATTTCCGGTATC 1301 CGCTCTCTGC GTTTCAGGCT TTCGCTATCT GTTTGAGCAG TTTCGACACA1351 AAACTTGCTT GCGAA AtTLP2: polypeptide:   1 MSLKSILRDL KEVRDGLGGISKRSWSKSSH IAPDQTTPPL DNIPQSPWAS (SEQ ID NO: 2)  51 LPPELLHDIIWRVEESETAW PARAAVVSCA SVCKSWRGIT MEIVRIPEQC  101 GKLTFPISLK QPGPRDSPIQCFIKRNRATA TYILYYGLMP SETENDKLLL  151 AARRIRRATC TDFIISLSAK NFSRSSSTYVGKLRSGFLGT KFTIYDNQTA  201 SSTAQAQPNR RLHPKQAAPK LPTNSSTVGN ITYELNVLRTRGPRRMHCAM  251 DSIPLSSVIA EPSVVQGIEE EVSSSPSPKG ETITTDKEIP DNSPSLRDQP 301 LVLKNKSPRW HEQLQCWCLN FKGRVTVASV KNFQLVAEID ASLDAPPEEH  351ERVILQFGKI GKDIFTMDYR YPLSAFQAFA ICISSFDTKP ACEG cDNA:   1 ATGTCTTTGAAAAGCATCCT TCGTGATCTG AAGGAAGTGA GGGATGGACT (SEQ ID NO: 13)  51TGGAGGCATC TCCAAGAGAA GCTGGTCAAA GTCGTCTCAC ATTGCTCCTG  101 ATCAAACAACTCCACCACTG GATAACATAC CACAGAGCCC ATGGGCTTCT  151 TTGCCGCCTG AGTTGCTTCATGACATTATC TGGAGGGTTG AAGAGAGTGA  201 GACAGCTTGG CCCGCTCGAG CTGCCGTTGTCTCTTGTGCT TCAGTATGTA  251 AATCATGGAG AGGAATCACT ATGGAGATTG TGAGGATCCCTGAGCAGTGT  301 GGGAAGCTCA CTTTTCCAAT CTCATTGAAA CAGCCGGGGC CTCGAGACTC 351 TCCAATTCAA TGTTTTATTA AGAGGAACAG AGCAACAGCT ACATACATTC  401TCTATTATGG TTTGATGCCT TCGGAGACTG AGAACGACAA ACTGTTGTTA  451 GCAGCAAGAAGGATTAGAAG AGCGACATGC ACAGACTTTA TAATCTCCCT  501 ATCTGCAAAG AACTTCTCACGGAGCAGCAG TACTTATGTT GGCAAATTAA  551 GGTCTGGTTT TCTGGGAACC AAGTTCACAATATATGACAA CCAAACAGCA  601 TCATCCACAG CACAAGCCCA ACCTAACCGA AGACTCCACCCGAAACAAGC  651 GGCTCCTAAA CTACCTACGA ATAGCTCTAC CGTAGGAAAC ATAACCTACG 701 AGCTCAATGT TCTTCGCACA AGGGGACCTA GAAGAATGCA CTGCGCTATG  751GATTCTATAC CCCTCTCTTC TGTTATTGCT GAACCGTCAG TAGTTCAAGG  801 CATAGAAGAGGAAGTCTCTT CCTCTCCTTC ACCAAAAGGA GAAACCATCA  851 CAACAGACAA AGAGATTCCTGATAATTCTC CAAGCTTAAG GGACCAACCG  901 CTAGTTCTCA AAAACAAATC CCCAAGATGGCATGAGCAGT TGCAGTGCTG  951 GTGCCTCAAC TTCAAGGGAA GAGTGACTGT GGCTTCAGTTAAGAATTTCC 1001 AGCTTGTTGC AGAGATTGAC GCTTCTTTGG ATGCGCCGCC TGAAGAACAT1051 GAGAGGGTGA TCTTACAGTT TGGCAAAATC GGTAAGGATA TTTTCACCAT 1101GGATTATCGC TACCCTCTAT CTGCTTTTCA AGCCTTTGCT ATATGCATTA 1151 GCAGCTTTGACACCAAACCG GCATGTGAAG GG AtTLP3: polypeptide:   1 MSFKSLIQDM RGELGSISRKGFDVRFGYGR SRSQRVVQDT SVPVDAFKQS (SEQ ID NO: 3)  51 CWASMPPELLRDVLMRIEQS EDTWPSRKNV VSCAGVCRNW REIVKEIVRV  101 PELSSKLTFP ISLKQPGPRGSLVQCYIMRN RSNQTYYLYL GLNQAASNDD  151 GKFLLAAKRF RRPTCTDYII SLNCDDVSRGSNTYIGKLRS NFLGTKFTVY  201 DAQPTNPGTQ VTRTRSSRLL SLKQVSPRIP SGNYPVAHISYELNVLGSRG  251 PRRMQCVMDA IPASAVEPGG TAPTQTELVH SNLDSFPSFS FFRSKSIRAE 301 SLPSGPSSAA QKEGLLVLKN KAPRWHEQLQ CWCLNFNGRV TVASVKNFQL  351VAAPENGPAG PEHENVILQF GKVGKDVFTM DYQYPISAFQ AFTICLSSFD  401 TKIACE cDNA:  1 ATGTCCTTCA AGAGTCTCAT TCAGGACATG AGAGGAGAGC TTGGGAGTAT (SEQ IDN0:14)  51 ATCCAGAAAG GGATTCGATG TCAGATTCGG GTATGGTAGA TCCAGGTCTC  101AACGTGTTGT TCAGGATACT TCTGTTCCTG TTGATGCTTT CAAGCAGAGC  151 TGCTGGGCTAGTATGCCTCC GGAGCTCCTG AGAGATGTTC TTATGAGGAT  201 TGAGCAATCC GAAGACACTTGGCCGTCTAG GAAAAATGTT GTTTCTTGCG  251 CTGGTGTCTG CAGGAACTGG CGAGAAATCGTCAAAGAGAT CGTCAGAGTT  301 CCTGAGCTTT CTAGCAAACT CACTTTTCCT ATCTCCCTCAAACAGCCGGG  351 TCCTAGAGGA TCACTTGTTC AATGCTATAT TATGAGAAAC CGCAGCAATC 401 AAACCTACTA TCTATACCTC GGGTTAAACC AAGCAGCTTC AAATGATGAT  451GGAAAGTTCC TTCTTGCTGC CAAGAGGTTT CGGAGGCCAA CTTGCACTGA  501 CTACATCATCTCCTTAAACT GCGATGATGT CTCTCGAGGA AGCAATACCT  551 ATATCGGAAA GCTTAGATCTAACTTTCTGG GGACCAAGTT CACTGTCTAT  601 GACGCTCAGC CGACGAATCC TGGAACTCAGGTTACCAGAA CCCGTTCAAG  651 CAGACTTCTC AGTTTGAAAC AAGTGAGCCC GAGAATTCCATCTGGCAACT  701 ATCCTGTAGC ACATATCTCA TATGAGCTTA ACGTCTTGGG TTCCAGAGGA 751 CCGAGGAGGA TGCAGTGTGT CATGGATGCC ATCCCTGCAT CAGCTGTAGA  801ACCTGGAGGA ACAGCTCCAA CTCAGACGGA ACTTGTCCAT AGCAATCTTG  851 ATAGTTTCCCCTCATTCTCC TTCTTCAGGT CGAAATCAAT TCGTGCAGAG  901 AGTCTCCCTT CTGGTCCATCATCTGCTGCT CAGAAGGAAG GACTGCTTGT  951 CCTGAAAAAC AAAGCGCCCA GATGGCACGAACAGCTCCAG TGCTGGTGCC 1001 TCAACTTCAA TGGGAGAGTC ACAGTTGCTT CCGTCAAAAACTTTCAGCTG 1051 GTAGCTGCTC CTGAGAATGG ACCTGCAGGA CCTGAGCACG AAAACGTGAT1101 TCTCCAGTTT GGAAAAGTCG GAAAAGATGT GTTCACAATG GATTATCAGT 1151ACCCTATCTC TGCCTTCCAG GCCTTCACCA TTTGCCTCAG CAGTTTCGAC 1201 ACCAAGATAGCATGTGAA AtTLP4: polypeptide:   1 MPPELLRDVL MRIERSEDTW PSRKNVVSCVGVCKNWRQIF KEIVNVPEVS (SEQ ID NO:4)  51 SKFTFPISLK QPGPGGSLVQ CYVKRNRSNQTFYLYLGGEA KIFCQSEPSD  101 IYLVPYSYRE THCVMDAISA SAVKPGGTAT TQTELDNFVSFRSPSGQKEG  151 VLVLKSKVPR LEEQSWCLDF NGSPENEPEN ENDIFQFAKV GNLHKLFSLY 201 EAEWIPLVRT SVFAVIARVC RDKKHTPSYE LKLALYFAKN SAILKKFVLR  251GYTREEDLLA LPVAN cDNA:   1 ATGCCTCCTG AGCTTCTGAG AGATGTTCTG ATGAGGATAGAGCGATCCGA (SEQ ID NO:15)  51 AGACACTTGG CCTTCTAGGA AGAATGTTGTTTCTTGTGTA GGTGTGTGTA  101 AGAACTGGCG ACAAATATTC AAAGAGATCG TTAACGTTCCTGAGGTTTCT  151 AGCAAATTCA CTTTTCCAAT CTCCTTGAAA CAGCCTGGTC CAGGAGGATC 201 ACTTGTTCAA TGCTATGTTA AGAGAAACCG TAGCAATCAA ACTTTCTATC  251TATACCTTGG AGGTGAAGCA AAAATATTTT GTCAGTCTGA ACCAACTGAT  301 ATTTATCTCGTTCCTTACAG TTACAGAGAG ACGCATTGCG TCATGGATGC  351 CATCTCTGCA TCAGCAGTAAAACCTGCAGG AACAGCTACA ACTCAGACAG  401 AACTCGATAA TTTCGTGTCA TTCAGGTCTCCTTCTGGTCA AAAGGAAGGA  451 GTGCTTGTTC TTAAGAGCAA AGTGCCTAGA TTGGAAGAACAGAGCTGGTG  501 TCTCGACTTC AATGGCTCTC CTGAGAACGA ACCTGAGAAT GAAAACGACA 551 TTTTCCAGTT TGCGAAAGTC GGAAACTTGC ACAAACTCTT CAGTTTATAT  601GACCCTGAAT GCATTCCTCT CGTTCGCACC TCAGTGTTTG CTGTCATTGC  651 TCGAGTTTGTAGAGATAAAA AGCATACACC ATCGTATGAA TTGAAACTTG  701 CATTGTACTT TGCAAAAAACTCTGCAATCC TCAAGAAATT CGTTCTCCGC  751 GGTTACACTC GAGAAGAAGA TTTACTCGCATTGCCCGTGG CTAAC AtTLP5: polypeptide:   1 MSFLSIVRDV RDTVGSFSRRSFDVRVSNGT THQRSKSHGV EAHIEDLIVI (SEQ ID NO: 5)  51 KNTRWANLPAALLRDVMKKL DESESTWPAR KQVVACAGVC KTWRLMCKDI  101 VKSPEFSGKL TFPVSLKQPGPRDGIIQCYI KRDKSNMTYH LYLSLSPAIL  151 VESGKFLLSA KRSRRATYTE YVISMDADNISRSSSTYIGK LKSNFLGTKF  201 IVYDTAPAYN SSQILSPPNR SRSFNSKKVS PKVPSGSYNIAQVTYELNLL  251 GTRGPRRMNC IMHSIPSLAL EPGGTVPSQP EFLQRSLDES FRSIGSSKIV 301 NHSGDFTRPK EEEGKVRPLV LKTKPPRWLQ PLRCWCLNFK GRVTVASVKN  351FQLMSAATVQ PGSGSDGGAL ATRPSLSPQQ PEQSNHDKII LHFGKVGKDM  401 FTMDYRYPLSAFQAFAISLS TFDTKLACE cDNA:   1 ATGTCGTTTC TGAGTATTGT TCGTGATGTTAGAGATACTG TAGGAAGCTT (SEQ ID NO: 16)  51 TTCGAGACGT AGTTTCGACGTGAGAGTATC TAATGGGACG ACTCATCAGA  101 GGAGTAAATC TCACGGTGTT GAGGCACATATTGAAGATCT TATTGTAATC  151 AAGAACACTC GTTGGGCTAA TTTACCGGCT GCGCTATTACGAGATGTGAT  201 GAAAAAGTTG GATGAAAGCG AGAGTACTTG GCCTGCACGT AAACAAGTCG 251 TTGCTTGTGC TGGTGTCTGC AAGACATGGA GACTAATGTG CAAAGATATT  301GTGAAAAGTC CTGAGTTCTC AGGCAAACTC ACATTTCCAG TTTCGTTGAA  351 ACAGCCCGGGCCTAGGGATG GAATCATACA ATGTTATATC AAAAGAGACA  401 AGTCTAACAT GACTTACCACCTTTACCTTT CTCTTAGTCC TGCCATACTT  451 GTTGAAAGTG GGAAGTTTCT TCTCTCGGCAAAGCGCTCAC GGAGAGCTAC  501 ATACACAGAG TATGTAATAT CAATGGATGC AGACAACATTTCAAGATCAA  551 GCAGCACTTA CATTGGCAAA CTGAAGTCTA ACTTTCTAGG GACAAAATTT 601 ATAGTATATG ATACGGCTCC TGCGTACAAC AGTAGCCAGA TATTGTCCCC  651ACCAAACCGG AGTCGTAGTT TCAACTCCAA GAAAGTGTCT CCCAAAGTCC  701 CTTCTGGAAGTTACAACATT GCTCAAGTTA CATACGAGCT GAACTTGCTT  751 GGAACCCGTG GACCTCGTAGAATGAACTGC ATTATGCACT CTATCCCCTC  801 CTTAGCTCTA GAACCCGGAG GTACTGTCCCTAGCCAACCT GAGTTTCTAC  851 AACGTTCCCT TGATGAATCT TTCCGCAGCA TCCGTTCCTCAAAGATAGTC  901 AACCACTCGG GAGATTTCAC CCGACCGAAA GAGGAAGAAG GAAAGGTGCG 951 ACCTTTGGTA CTGAAAACTA AACCGCCAAG GTGGCTCCAA CCGTTGCGAT 1001GTTGGTGCCT TAACTTCAAA GGGAGAGTGA CTGTAGCTTC TGTCAAGAAC 1051 TTCCAGTTGATGTCCGCTGC AACGGTTCAG CCCGGTAGTG GTAGTGATGG 1101 TGGAGCATTG GCTACGAGGCCATCGTTATC ACCACAGCAG CCAGAGCAAT 1151 CAAACCATGA TAAGATAATA CTACACTTTGGGAAAGTGGG TAAGGATATG 1201 TTCACTATGG ACTATCGTTA TCCTCTCTCT GCCTTTCAAGCGTTTGCCAT 1251 TTCCCTGAGC ACCTTTGATA CTAAATTGGC ATGTGAA AtTLP6:polypeptide:   1 MSLKNIVKNK YKAIGRRGRS HIAPEGSSVS SSLSTNEGLN QSIWVDLPPE(SEQ ID NO:6)  51 LLLDIIQRIE SEQSLWPGRR DVVACASVCK SWREMTKEVV KVPELSGLIT 101 FPISLRQPGP RDAPIQCFIK RERATGIYRL YLGLSPALSG DKSKLLLSAK  151RVRRATGAEF VVSLSGNDFS RSSSNYIGKL RSNFLGTKFT VYENQPPPFN  201 RKLPPSMQVSPWVSSSSSSY NIASILYELN VLRTRGPRRM QCIMHSIPIS  251 AIQEGGKIQS PTEFTNQGKKKKKPLMDFCS GNLGGESVIK EPLILKNKSP  301 RWHEQLQCWC LNFKGRVTVA SVKNFQLVAAAAEAGKNMNI PEEEQDRVIL  351 QFGKIGKDIF TMDYRYPISA FQAFAICLSS FDTKPVCEcDNA:   1 ATGTCATTGA AGAACATAGT GAAGAACAAA TACAAAGCTA TTGGTAGAAG (SEQ IDNO:17)  51 AGGGAGGTCA CACATTGCAC CAGAAGGATC ATCTGTGTCT TCTTCTTTAT  101CAACTAATGA AGGTTTAAAC CAGAGTATTT GGGTTGATTT GCCTCCAGAG  151 TTACTTCTTGATATAATCCA AAGGATTGAG TCTGAACAGA GTTTATGGCC  201 GGGGAGGAGA GATGTTGTTGCTTGTGCTTC GGTTTGTAAG TCATGGAGGG  251 AGATGACTAA AGAAGTTGTT AAAGTTCCTGAGCTCTCTGG TTTGATCACG  301 TTTCCGATTT CTTTAAGACA GCCTGGACCT AGAGATGCTCCAATTCAATG  351 CTTTATTAAA CGTGAAAGAG CTACGGGGAT ATACCGTCTC TATCTTGGTT 401 TAAGCCCTGC TCTTTCCGGT GACAAGAGTA AGTTGTTGTT ATCTGCAAAG  451AGAGTCAGGA GAGCGACGGG TGCGGAGTTT GTTGTATCGT TATCGGGGAA  501 TGACTTCTCGAGAAGTAGTA GTAATTACAT AGGAAAACTG AGATCAAATT  551 TCCTGGGAAC GAAGTTCACAGTCTACGAAA ACCAACCTCC TCCGTTTAAC  601 CGAAAGCTCC CACCATCGAT GCAAGTGTCTCCATGGGTAT CGTCGTCATC  651 TAGTAGTTAC AACATAGCTT CAATCTTGTA TGAGCTGAATGTTCTGAGAA  701 CCAGAGGTCC AAGAAGAATG CAATGTATAA TGCACAGTAT CCCGATTTCA 751 GCGATTCAAG AAGGCGGCAA AATCCAGTCG CCAACGGAGT TCACAAACCA  801AGGAAAGAAG AAGAAGAAGC CGCTGATGGA TTTCTGCTCA GGGAACCTGG  851 GAGGAGAATCCGTTATAAAA GAACCATTAA TTCTGAAAAA CAAGTCGCCG  901 AGATGGCACG AACAGCTTCAGTGCTGGTGT CTAAACTTCA AAGGTCGAGT  951 CACAGTCGCC TCGGTGAAAA ACTTCCAGCTAGTGGCAGCT GCTGCAGAAG 1001 CAGGGAAGAA CATGAACATA CCAGAAGAGG AACAAGATAGAGTGATATTA 1051 CAGTTTGGGA AGATAGGCAA AGACATTTTC ACAATGGATT ATCGTTACCC1101 GATCTCTGCA TTCCAAGCTT TTGCTATTTG TTTAAGCAGC TTCGACACGA 1151AGCCAGTCTG CGAA AtTLP7: polypeptide:   1 MPLSRSLLSR RISNSFRFHQGETTTAPESE SIPPPSNMAG SSSWSAMLPE (SEQ ID NO:7)  51 LLGEIIRRVE ETEDRWPQRRDVVTCACVSK KWREITHDFA RSSLNSGKIT  101 FPSCLKLPGP RDFSNQCLIK RNKKTSTFYLYLALTPSFTD KGKFLLAARR  151 FRTGAYTEYI ISLDADDFSQ GSNAYVGKLR SDFLGTNFTVYDSQPPHNGA  201 KPSNGKASRR FASKQISPQV PAGNFEVGHV SYKFNLLKSR GPRRMVSTLR 251 CPSPSPSSSS AGLSSDQKPC DVTKIMKKPN KDGSSLTILK NKAPRWHEHL  301QCWCLNFHGR VTVASVKNFQ LVATVDQSQP SGKGDEETVL LQFGKVGDDT  351 FTMDYRQPLSAFQAFAICLT SFGTKLACE cDNA:   1 ATGCCTTTGT CACGGTCCCT CCTTTCGCGGAGGATCTCGA ACTCTTTTAG (SEQ ID NO:18)  51 GTTTCATCAG GGAGAGACAACGACGGCACC GGAATCCGAA TCGATTCCTC  101 CGCCGTCGAA TATGGCCGGT TCTTCGTCATGGTCGGCGAT GCTCCCTGAA  151 TTATTAGGCG AGATCATTCG TCGCGTGGAG GAGACTGAGGACCGTTGGCC  201 TCAACGTCGT GATGTAGTTA CTTGCGCTTG CGTTTCTAAG AAATGGAGAG 251 AAATCACTCA CGATTTCGCT AGATCCTCTC TTAACTCTGG CAAAATTACT  301TTCCCTTCTT GCCTCAAATT GCCAGGTCCT AGAGACTTTT CTAATCAGTG  351 CTTGATAAAGAGGAACAAGA AGACATCAAC GTTTTACTTG TATCTTGCTC  401 TAACACCATC ATTCACTGATAAGGGAAAGT TTCTTCTGGC GGCGCGOAGG  451 TTTAGGACCG GTGCTTACAC TGAGTACATCATATCACTTG ATGCTGATGA  501 TTTCTCTCAA GGAAGTAATG CCTACGTCGG AAAATTAAGATCAGATTTTC  551 TTGGGACCAA CTTTACAGTA TACGATAGCC AACCACCACA CAACGGAGCA 601 AAACCTTCAA ATGGCAAAGC CAGTCGCAGA TTTGCATCAA AGCAGATAAG  651CCCTCAAGTT CCAGCAGGCA ACTTTGAAGT CGGTCATGTT TCTTATAAAT  701 TCAACCTTTTGAAATCAAGA GGTCCAAGAA GAATGGTAAG CACACTCCGA  751 TGCCCATCAC CATCACCTTCATCATCATCC GCTGGACTCT CGTCTGACCA  801 AAAGCCATGT GATGTAACCA AGATAATGAAAAAACCCAAC AAGGATGGTT  851 CCAGCTTGAC AATACTAAAG AACAAAGCTC CTAGATGGCACGAGCACTTG  901 CAGTGCTGGT GTCTGAACTT CCATGGACGA GTTACTGTTC CTTCGGTCAA 951 GAACTTTCAG CTGGTTGCGA CCGTTGACCA AAGTCAACCG AGCGGTAAAG 1001GCGATGAAGA AACAGTTCTT CTACAGTTTG GTAAAGTGGG AGATGACACT 1051 TTCACTATGGATTATAGACA GCCTCTCTCT GCATTTCAGG CTTTTGCTAT 1101 CTGTCTCACA AGTTTCGGCACTAAACTTGC CTGCGAG AtTLP8: polypeptide:   1 MAGSRKVNDL LEENKGNVDTITGSLSTQKG EDKENVSPEK VSTSVETRKL (SEQ ID NO:8)  51 DRALKSQSMK GNSGFPTEVTNFKSFSTGGR TALKQSSLQA CMQKNSEVDK  101 SSFGMKTWTS VDSEHSSSLK VWEFSDSEAAPASSWSTLPN RALLCKTLPL  151 DVGRCTCLIV KEQSPEGLSH GSVYSLYTHE GRGRKDRKLAVAYHSRRNGK  201 SIFRVAQNVK GLLCSSDESY VGSMTANLLG SKYYIWDKGV RVGSVGKMVK 251 PLLSVVIFTP TITTWTGSYR RMRTLLPKQQ PMQKNNNKQV QQASKLPLDW  301LENKEKIQKL CSRIPHYNKI SKQHELDFRD RGRTGLRIQS SVKNFQLTLT  351 ETPRQTILQMGRVDKARYVI DFRYPFSGYQ AFCICLASID SKLCCTV cDNA:   1 ATGGCTGGTT CGAGAAAAGTGAATGATTTG TTGGAGGAAA ATAAGGGAAA (SEQ ID NO:19)  51 TGTGGACACAATTACAGGGT CTTTATCCAC TCAAAAGGGA GAGGATAAGG  101 AGAATGTGTC GCCGGAGAAAGTCTCTACCT CTGTGGAAAC TCGGAAACTA  151 GATCGAGCTT TGAAGTCTCA ATCGATGAAGGGTAACTCTG GGTTTCCAAC  201 GGAAGTTACA AATTTCAAAT CTTTCTCAAC TGGTGGTCGAACAGCTCTGA  251 AGCAGTCATC ACTGCAAGCG TGTATGCAGA AGAACAGTGA GGTTGATAAG 301 AGTAGTTTCG GAATGAAAAC TTGGACTAGT GTTGATTCAG AGCATTCAAG  351TTCGTTGAAA GTGTGGGAGT TTTCGGATTC TGAAGCTGCC CCTGCTTCCT  401 CTTGGTCTACTTTGCCCAAC AGGGCTTTGT TGTGCAAGAC ACTACCTTTG  451 GATGTGGGAA GATGCACTTGTCTGATTGTG AAAGAACAAT CACCTGAAGG  501 CTTGAGCCAC GGATCTGTAT ATTCACTTTATACACATGAA GGTCGGGGGC  551 GTAAAGACCG GAAGTTAGCA GTTGCTTACC ATAGCCGACGTAATGGGAAA  601 TCTATATTTA GGGTGGCACA GAATGTTAAG GGATTGCTGT GCAGTTCGGA 651 TGAAAGTTAT GTCGGTTCCA TGACGGCTAA TCTCTTGGGT TCCAAGTACT  701ACATATGGGA CAAGGGAGTT CGAGTTGGTT CTGTAGGTAA AATGGTGAAG  751 CCGCTTCTTTCGGTTGTAAT ATTCACACCC ACCATAACAA CTTGGACAGG  801 GAGCTACAGA AGAATGAGAACTTTGCTACC AAAGCAGCAG CCAATGCAGA  851 AAAACAACAA TAAGCAGGTT CAACAAGCTAGTAAACTACC GCTTGATTGG  901 CTTGAGAATA AGGAAAAAAT TCAGAAGCTA TGCTCAAGGATACCACATTA  951 CAACAAAATC TCCAAGCAGC ATGAGTTAGA CTTCAGAGAC AGAGGAAGAA1001 CAGGACTGAG AATACAGAGC TCGGTGAAGA ACTTTCAGCT AACACTCACG 1051GAGACTCCAA GGCAGACAAT TCTTCAAATG GGGAGAGTTG ACAAAGCAAG 1101 ATATGTAATCGACTTCAGGT ATCCATTCTC AGGCTACCAA GCATTCTGCA 1151 TTTGCTTGGC TTCTATTGATTCCAAGCTTT GTTGTACTGT T AtTLP9: polypeptide:   1 MTFRSLLQEM RSRPHRVVHAAASTANSSDP FSWSELPEEL LREILIRVET (SEQ ID NO:9)  51 VDGGDWPSRR NVVACAGVCRSWRILTKEIV AVPEFSSKLT FPISLKQSGP  101 RDSLVQCFIK RNRNTQSYHL YLGLTTSLTDNGKFLLAASK LKRATCTDYI  151 ISLRSDDISK RSNAYLGRMR SNFLGTKFTV FDGSQTGAAKMQKSRSSNFI  201 KVSPRVPQGS YPIAHISYEL NVLGSRGPRR MRCIMDTIPM SIVESRGVVA 251 STSISSFSSR SSPVFRSHSK PLRSNSASCS DSGNNLGDPP LVLSNKAPRW  301HEQLRCWCLN FHGRVTVASV KNFQLVAVSD CEAGQTESRI ILQFGKVGKD  351 MFTMDYGYPISAFQAFAICL SSFETRIACE cDNA:   1 ATGACGTTCC GAAGTTTACT CCAGGAAATGCGGTCTAGGC CACACCGTGT (SEQ ID NO:20)  51 AGTTCACGCC GCCGCCTCAACCGCTAATAG TTCAGACCCT TTCAGCTGGT  101 CGGAGCTCCC GGAGGAGCTG CTTAGAGAAATCCTGATTAG GGTTGAGACT  151 GTTGACGGCG GCGATTGGCC GTCGCGGCGA AACGTGGTGGCTTGTGCCGG  201 CGTTTGTCGT AGCTGGAGGA TTCTCACCAA GGAGATTGTA GCTGTTCCTG 251 AATTCTCCTC TAAATTGACT TTCCCTATCT CCCTCAAGCA GTCTGGTCCA  301AGAGATTCTC TAGTTCAATG CTTTATAAAA CGTAATCGAA ATACTCAATC  351 GTATCATCTCTATCTCGGAT TAACTACCTC TTTGACGGAT AACGGGAAGT  401 TTCTTCTTGC TGCTTCTAAGCTGAAGCGCG CAACTTGCAC TGATTACATC  451 ATCTCTTTGC GTTCAGACGA TATCTCAAAGAGAAGCAACG CGTATCTTGG  501 GAGAATGAGA TCGAACTTCC TTGGAACAAA ATTCACGGTCTTTGATGGTA  551 GTCAGACCGG AGCAGCGAAG ATGCAGAAGA GCCGCTCTTC TAATTTCATC 601 AAAGTTTCAC CTAGAGTTCC TCAGGGAAGT TACCCCATCG CTCACATTTC  651ATACGAGTTA AACGTCTTAG GCTCTCGGGG ACCGAGAAGA ATGCGTTGCA  701 TCATGGATACAATACCTATG AGCATCGTGG AGTCGCGAGG AGTAGTAGCT  751 TCAACATCCA TAAGCTCTTTTTCCAGTCGG TCATCACCAG TCTTTAGGTC  801 TCACTCAAAA CCATTGCGCA GTAATAGTGCATCATGTAGC GACTCAGGCA  851 ACAACCTGGG AGATCCACCA TTGGTGCTGA GCAACAAAGCTCCACGGTGG  901 CATGAGCAGT TACGTTGCTG GTGCTTAAAT TTCCATGGTC GAGTCACAGT 951 GGCTTCGGTT AAGAACTTTC AGCTTGTGGC AGTTAGTGAC TGTGAAGCAG 1001GGCAGACATC TGAGAGGATC ATACTCCAGT TTGGGAAAGT TGGGAAGGAC 1051 ATGTTTACCATGGATTATGG ATATCCGATT TCTGCGTTTC AAGCGTTTGC 1101 TATCTGCCTG AGCAGTTTTGAAACCAGAAT TGCCTGTGAA AtTLP10: polypeptide:   1 MSFRGIVQDL RDGFGSLSRRSFDFRLSSLH KGKAQGSSFR EYSSSRDLLS (SEQ ID NO:10)  51 PVIVQTSRWANLPPELLFDV IKRLEESESN WPARKHVVAC ASVCRSWRAM  101 CQEIVLGPEI CGKLTFPVSLKQPGPRDAMI QCFIKRDKSK LTFHLFLCLS  151 PALLVENGKF LLSAKRTRRT TRTEYIISMDADNISRSSNS YLGKLRSNFL  201 GTKFLVYDTQ PPPNTSSSAL ITDRTSRSRF HSRRVSPKVPSGSYNIAQIT  251 YELNVLGTRG PRRMHCIMNS IPISSLEPGG SVPNQPEKLV PAPYSLDDSF 301 RSNISFSKSS FDHRSLDFSS SRFSEMGISC DDNEEEASFR PLILKNKQPR  351WHEQLQCWCL NFRGRVTVAS VKNFQLVAAR QPQPQGTGAA AAPTSAPAHP  401 EQDKVILQFGKVGKDMFTMD YRYPLSAFQA FAICLSSFDT KLACE cDNA:   1 ATGTCGTTTC GAGGCATTGTTCAAGATTTG AGAGATGGGT TTGGGAGCTT (SEQ ID NO:21)  51 GTCAAGGAGGAGTTTCGATT TTAGGCTCTC GAGTCTTCAT AAAGGGAAAG  101 CTCAGGGTTC TTCGTTCCGTGAGTATTCGT CATCCCGTGA TCTCTTGTCG  151 CCTGTGATAG TTCAGACAAG TAGATGGGCTAATCTTCCTC CAGAGTTACT  201 CTTTGATGTG ATCAAAAGAT TAGAGGAAAG TGAGAGTAATTGGCCTGCAA  251 GAAAACATGT TGTGGCTTGT GCTTCGGTTT GTCGGTCTTG GAGAGCTATG 301 TGCCAAGAGA TTGTTTTGGG GCCTGAAATC TGTGGGAAAC TCACTTTCCC  351TGTTTCCCTC AAACAGCCAG GGCCTCGTGA TGCAATGATT CAGTGTTTCA  401 TCAAAAGGGATAAATCAAAG CTAACATTTC ACCTTTTTCT TTGTTTAAGT  451 CCCGCTCTAT TAGTGGAGAATGGGAAATTT CTTCTTTCAG CTAAAAGAAC  501 TCGTAGAACT ACTCGAACCG AGTACATTATCTCCATGGAT GCTGATAACA  551 TCTCAAGATC CAGCAACTCT TACCTCGGAA AGCTCAGATCAAACTTCCTT  601 GGGACAAAGT TCTTGGTGTA CGACACGCAA CCACCACCAA ACACATCTTC 651 GAGCGCACTT ATCACTGATC GAACAAGCCG AAGCAGGTTT CACTCCAGAC  701GAGTTTCTCC TAAAGTACCA TCCGGAAGCT ACAACATTGC TCAAATCACC  751 TATGAGCTCAACGTGTTGGG CACACGCGGG CCACGACGAA TGCACTGCAT  801 CATGAACTCC ATCCCAATTTCATCGCTCGA ACCAGGCGGT TCAGTCCCTA  851 ACCAACCCGA GAAACTCGTC CCTGCACCATACTCTCTCGA CGACTCATTC  901 CGCAGTAACA TCTCCTTCTC CAAATCATCA TTTGACCACCGCTCCCTCGA  951 TTTCAGCAGT TCTAGATTCT CCGAAATGGG AATATCCTGC GACGACAACG1001 AAGAAGAAGC GAGTTTCAGA CCGTTGATTC TAAAGAACAA GCAGCCAAGG 1051TGGCACGAGC AGTTGCAATG CTGGTGTTTG AATTTCCGCG GACGTGTGAC 1101 AGTTGCATCGGTTAAGAATT TCCAGCTTGT AGCAGCAAGA CAGCCGCAGC 1151 CTCAAGGGAC AGGTGCAGCAGCAGCACCAA CAAGTGCACC TGCTCACCCT 1201 GAGCAAGACA AGGTGATTCT CCAGTTTGGTAAAGTAGGGA AAGATATGTT 1251 CACAATGGAC TATAGGTATC CATTATCGGC GTTTCAGGCGTTTGCGATAT 1301 GCTTAAGCAG CTTTGACACC AAGCTTGCTT GTGAA AtTLP11:polypeptide:   1 MRSRPHRVVH DLAAAAAADS TSVSSQDYRW SEIPEELLRE ILIRVEAADG(SEQ ID NO:11)  51 GGWPSRRSVV ACAGVCRGWR LLMNETVVVP EISSKLTFPISLKQPGPRDS  101 LVQCFIKRNR ITQSYHLYLG LTNSLTDDGK FLLAACKLKH TTCTDYIISL 151 RSDDMSRRSQ AYVGKVRSNF LGTKFTVFDG NLLPSTGAAK LRKSRSYNPA  201KVSAKVPLGS YPVAHITYEL NVLGSRGPRK MQCLMDTIPT STMEPQGVAS  251 EPSEFPLLGTRSTLSRSQSK PLRSSSSHLK ETPLVLSNKT PRWHEQLRCW  301 CNLFHGRVTV ASVKNFQLVAAGASCGSGTG MSPERQSERI ILQFGKVGKD  351 MFTMDYGYPI SAFQAFAICL SSFETRIACEcDNA:   1 ATGCGTTCGA GACCGCATCG TGTGGTCCAC GACCTTGCCG CCGCCGCAGC (SEQ IDNO:22)  51 TGCCGATTCC ACTTCTGTGT CATCGCAAGA TTATCGCTGG TCAGAGATTC  101CTGAAGAGCT TCTTAGGGAG ATTCTGATTC GTGTTGAAGC GGCGGACGGT  151 GGCGGATGGCCGTCACGACG CAGCGTGGTG GCTTGTGCCG GCGTTTGTCG  201 TGGCTGGCGG CTACTTATGAACGAAACCGT CGTTGTCCCT GAGATCTCTT  251 CTAAGTTGAC TTTCCCCATC TCTCTCAAGCAGCCTGGTCC AAGGGATTCA  301 CTGGTTCAAT GCTTTATCAA ACGTAATCGA ATTACGCAATCATATCATCT  351 CTATCTCGGA TTAACCAACT CTTTAACGGA TGATGGGAAG TTTTTGCTTG 401 CTGCGTGTAA GTTGAAGCAC ACAACTTGTA CGGATTACAT TATCTCTTTA  451CGTTCTGATG ATATGTCGAG AAGAAGCCAA GCTTATGTTG GCAAAGTGAG  501 ATCGAACTTCCTAGGAACGA AATTCACTGT CTTTGATGGA AATCTGCTGC  551 CTTCAACGGG AGCCGCAAAGTTGAGAAAGA GCCGATCTTA TAATCCCGCA  601 AAAGTTTCAG CAAAAGTTCC TCTTGGAAGTTATCCTGTCG CTCATATCAC  651 ATATGAGCTG AATGTCTTAG GATCCCGGGG ACCAAGAAAGATGCAATGTC  701 TTATGGACAC AATACCTACA AGCACAATGG AGCCTCAAGG AGTAGCTTCA 751 GAACCATCAG AGTTTCCCTT ACTCGGTACT CGGTCAACCT TATCCAGGTC  801TCAGTCAAAA CCATTACGCA GTAGCTCAAG CCACCTGAAA GAAACACCAT  851 TAGTGCTGAGCAACAAGACA CCACGGTGGC ACGAGCAGCT ACGCTGCTGG  901 TGCTTGAATT TCCATGGCCGTGTCACAGTA GCGTCAGTGA AGAACTTTCA  951 GCTCGTGGCA GCAGGAGCTA GCTGTGGCAGTGGCACGGGA ATGTCACCGG 1001 AGAGGCAGAG CGAGCGGATT ATATTGCAGT TTGGGAAAGTCGGGAAAGAT 1051 ATGTTCACGA TGGATTATGG ATACCCGATC TCAGCTTTCC AGGCTTTTGC1101 CATTTGCTTG AGCAGCTTTG AGACTAGAAT CGCTTGTGAA

Accordingly, one aspect of the invention features an isolatedpolypeptide containing an amino acid sequence at least 70% (i.e., anynumber between 70% and 100%, inclusive) identical to one of SEQ ID NOs:1-11. When expressed in a plant cell, e.g., an Arabidopsis cell, thepolypeptide regulates the transcription of genes, in response toenvironmental stimuli. The polypeptide of the invention can be used toidentify DNA elements, such as promoters, enhances, or silencers, whichit binds to. Such DNA elements mediate the response of plants to variousenvironmental factors. The polypeptide of the invention can also be usedfor producing anti-AtTLP antibodies (either monoclonal or polyclonal).These antibodies in turn are useful for detecting the presence anddistribution of AtTLP proteins in tissues and in cellular compartments.For example, such antibodies can be used to verify the expression of TLPproteins in a transgenic plant.

An isolated polypeptide refers to a polypeptide substantially free fromnaturally associated molecules, i.e., it is at least 75% (i.e., anynumber between 75% and 100%, inclusive) pure by dry weight. Purity canbe measured by any appropriate standard method, for example, by columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

The percent identity of two amino acid sequences is determined using thealgorithm of Karlin and Altschul ((1990) Proc. Natl. Acad. Sci. USA 87,2264-2268), modified as in Karlin and Altschul ((1993) Proc. Natl. Acad.Sci. USA 90, 5873-5877). Such an algorithm is incorporated into theXBLAST programs of Altschul, et al. ((1990) J. Mol. Biol. 215, 403-410).BLAST protein searches are performed with the XBLAST program, score=50,wordlength=3. Where gaps exist between two sequences, Gapped BLAST isutilized as described in Altschul, et al. ((1997) Nucleic Acids Res. 25,3389-3402). When employing BLAST and Gapped BLAST programs, one canconveniently use the default parameters (e.g., XBLAST). Seencbi.nlm.nih.gov.

The invention further features (1) an isolated nucleic acid encoding apolypeptide of the invention and (2) an isolated nucleic acid that,under a high stringent condition, hybridizes to a probe containing asequence selected from the group consisting of SEQ ID NOs: 12-22, or acomplement of any selected sequence. Such a nucleic acid is at least 15(e.g., at least 30, 50, 100, 200, 500, or 1000) nucleotides in length.By hybridization under a high stringent condition is meant hybridizationat 65° C., 0.5×SSC, followed by washing at 45° C., 0.1×SSC. The nucleicacids of the invention can be used to determine whether an AtTLP mRNA isexpressed in a tissue or cell. The nucleic acids can be used as primersin PCR-based detection methods, or as labeled probes in nucleic acidblots (e.g., Northern blots).

An isolated nucleic acid refers to a nucleic acid the structure of whichis not identical to that of any naturally occurring nucleic acid or tothat of any fragment of a naturally occurring genomic nucleic acid. Theterm therefore covers, for example, (a) a DNA which has the sequence ofpart of a naturally occurring genomic DNA molecule but is not flanked byboth of the coding sequences that flank that part of the molecule in thegenome of the organism in which it naturally occurs; (b) a nucleic acidincorporated into a vector or into the genomic DNA of a prokaryote oreukaryote in a manner such that the resulting molecule is not identicalto any naturally occurring vector or genomic DNA; (c) a separatemolecule such as a cDNA, a genomic fragment, a fragment produced bypolymerase chain reaction (PCR), or a restriction fragment; and (d) arecombinant nucleotide sequence that is part of a hybrid gene, i.e., agene encoding a fusion protein.

The invention also features a vector and a host cell containing anucleic acid of the invention. The host cell can be an E. coli., ayeast, an insect, a plant (e.g., Arabidopsis), or a mammalian cell. Thevector and host cell can be used for producing a polypeptide of theinvention. For this purpose, one can culture the host cell in a mediumunder conditions permitting expression of the polypeptide, and isolatethe polypeptide.

The just-described vector and host cell can also be used for generatinga transformed plant cell or a transgenic plant containing a recombinantnucleic acid that encodes a heterologous polypeptide of SEQ ID NO: 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or 11. One can generate such a transformedplant cell by introducing into a plant cell a recombinant nucleic acidencoding such a heterologous polypeptide and expressing the polypeptidein the cell. To generate a transgenic plant, one can (1) introduce intoa plant cell a recombinant nucleic acid encoding one just-describedheterologous polypeptide; (2) expressing the polypeptide in the cell,and (3) cultivating the cell to generate a plant. The transformed plantcell or transgenic plant is more sensitive to environmental factors,such as high salinity, pathogens, and chilling, and therefore can beused as a sensor to detect and monitor small changes in environment,such as soil and air.

Also within the scope of this invention are a homozygous transformedplant cell (e.g., an Arabidopsis cell) and a transgenic plant (e.g.Arabidopsis) that lack a polypeptide containing a sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. The transformed plant cell ortransgenic plant, compared with the wild type cell or plant, has ahigher (by at least 30%, e.g., 50%, 90%, 100%, 200%) tolerance to salt,chilling, pathogens, oxidative stress, or water-deficit due to absenceof or lowered level of the polypeptide. In addition, the inventionfeatures method of making the transformed plant cell and the transgenicplant. Both methods include introducing into a plant cell a nucleic acid(e.g., a T-DNA, an anti-sense RNA, and an iRNA) that decreases theexpression of a gene encoding a polypeptide of SEQ ID NO: 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or 11. The method for making the plant further includescultivating the plant cell to generate a plant.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DETAILED DESCRIPTION

This invention is based on an unexpected discovery that (1)overexpression of AtTLPs in Arabidopsis increases sensitivity of theplant to various environmental factors, such as salt, chilling,oxidative stress, or water-deficit; and (2) lack of expression of AtTLPsincreases tolerance of the plant to several environmental factors.

Accordingly, in one aspect, the invention features a transformed plantcell containing a recombinant nucleic acid that encodes a heterologousAtTLP. The AtTLP proteins useful for this invention include, ArabidopsisAtTLPs 1-11 and TLPs of other species. The plant cell can be a dicotplant cell (e.g., a tomato cell, a brassica cell, or a potato cell) or amonocot plant cell (e.g. a rice cell, a wheat cell, or a barley cell).

A transformed plant cell of the invention can be produced by introducinginto a plant cell a recombinant nucleic acid that encodes a heterologousAtTLP protein and expressing the protein in the cell. Techniques fortransforming a wide variety of plant cells are well known in the art andcan be found in technical and scientific literature. See, for example,Weising et al., 1988, Ann. Rev. Genet. 22:421-477. To express aheterologous AtTLP gene in a plant cell, the gene can be combined withtranscriptional and translational initiation regulatory sequences thatdirect the transcription of the gene and translation of the encodedprotein in the plant cell.

For overexpression, a constitutive plant promoter may be employed. Aconstitutive promoter is active under most environmental conditions andstates of cell differentiation. Examples of constitutive promotersinclude the cauliflower mosaic virus (CaMV) 35S transcription initiationregion, the 1′- or 2′-promoter derived from T-DNA of Agrobacteriumtumafaciens, the ACT11 and Cat3 promoters from Arabidopsis (Huang etal., 1996, Plant Mol. Biol. 33:125-139 and Zhong et al., 1996, Mol. Gen.Genet. 251:196-203), the stearoyl-acyl carrier protein desaturase genepromoter from Brassica napus (Solocombe et al., 1994, Plant Physiol.104:1167-1176), and the GPc1 and Gpc2 promoters from maize (Martinez etal., 1989, J. Mol. Biol. 208:551-565 and Manjunath et al., 1997, PlantMol. Biol. 33:97-112).

Alternatively, a tissue-specific promoter or an inducible promoter maybe employed to direct expression of the AtTLP gene in a specific celltype or under more precise environmental or developmental control.Examples of environmental conditions that may affect transcription byinducible promoters include anaerobicity, elevation of temperature,presence of light, spray with chemicals or hormones, or infection by apathogen. Examples of a tissue-specific promoter or an induciblepromoter include the root-specific ANR1 promoter (Zhang and Forde, 1998,Science 279:407) and the photosynthetic organ-specific RBCS promoter(Khoudi et al., 1997, Gene 197:343).

For proper polypeptide expression, a polyadenylation region at the3′-end of the coding region should be included. The polyadenylationregion can be derived from the same gene, from a variety of other genes,or from T-DNA.

A marker gene can also be included to confer a selectable phenotype onplant cells. For example, the marker gene may encode a protein thatconfers biocide resistance, antibiotic resistance (e.g., resistance tokanamycin, G418, bleomycin, hygromycin), or herbicide resistance (e.g.,resistance to chlorosulfuron or Basta).

A recombinant nucleic acid that encodes a heterologous AtTLP protein maybe introduced into the genome of a desired plant host cell by a varietyof conventional techniques. For example, the recombinant nucleic acidmay be introduced directly into the genomic DNA of a plant cell usingtechniques such as polyethylene glycol precipitation, electroporation,microinjection, or ballistic methods (e.g., DNA particle bombardment).See, e.g., Paszkowski et al., 1984, EMBO J. 3:2717-2722, Fromm et al.,1985, Proc. Natl. Acad. Sci. USA 82:5824, and Klein et al., 1987, Nature327:70-73. Alternatively, the recombinant nucleic acid may be combinedwith suitable T-DNA flanking regions and introduced into a conventionalAgrobacterium tumefaciens host vector. The virulence functions of theAgrobacterium tumefaciens host direct the insertion of the AtTLP geneand adjacent marker into the plant cell DNA when the cell is infected bythe bacteria. Agrobacterium tumefaciens-mediated transformationtechniques, including disarming and use of binary vectors, are wellknown in the art. See, e.g., Horsch et al., 1984, Science 233:496-498;Fraley et al., 1983, Proc. Natl. Acad. Sci. USA 80:4803; and GeneTransfer to Plants, Potrykus, ed., Springer-Verlag, Berlin, 1995.

The presence and copy number of a heterologous AtTLP gene in atransgenic plant can be determined using standard methods, e.g.,Southern blotting. Expression of the heterologous AtTLP gene in atransgenic plant can be confirmed by detecting and quantifying theheterologous AtTLP mRNA or protein in the transgenic plant.

The transformed plant cells thus obtained can then be cultured toregenerate a whole plant. Regeneration techniques rely on manipulationof certain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide or herbicide marker that has been introducedtogether with a heat shock factor gene. Plant regeneration from culturedprotoplasts is described in Evans et al., Protoplasts Isolation andCulture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee etal., 1987, Ann. Rev. Plant Phys. 38:467-486. Once the heterologous AtTLPgene has been confirmed to be stably incorporated in the genome of atransgenic plant, it can be introduced into other plants by sexualcrossing. Depending upon the species to be crossed, one or more standardbreeding techniques can be used to generate the whole plant.

In another aspect, the invention feature a homozygous transformed plantcell that lack one or more of AtTLPs 1-11. Absence of the AtTLP(s)enhances tolerance of the cell to various environmental factors, e.g.,high salinity. Such a transformed cell can be made by introducing into aplant cell a nucleic acid that lowers the expression of a gene encodinga polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. Thenucleic acid, e.g., T-DNA, antisense RNA, or iRNA, can be introducedinto the cell using one of the standard transforming techniquesdescribed above. Stable transformants can be selected using the markergenes and selection methods also described above. A whole plant can thenbe regenerated from the transformed plant cells. It can be furthercrossed using conventional breeding techniques to generate homozygousplant.

The specific example below is to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentinvention to its fullest extent. All publications cited herein arehereby incorporated by reference in their entirety.

Identification of the AtTLP Family

A tubby consensus sequence (Pfam PF01167, Kleyn et al., 1996, Cell 85:281-290 and Noben-Trauth et al., 1996, Nature 380: 534-538) was used tosearch the Arabidopsis thaliana expressed sequence tag (EST) databaseand the completed Arabidopsis genome sequence (The Institute of GenomeResearch, TIGR) with multiple BLAST algorithms to locate all thesequences sharing significant similarities with the tubby domain(P-value <0.0085). The search results revealed eleven TUBBY-like proteingenes, termed AtTLP1 to AtTLP11, in the Arabidopsis genome. For each ofthe 11 genes, the corresponding BAC locus (The Arabidopsis InformationResource), Tentative Consensus (TC) group, AGI gene code, cDNA GenBankaccession number, and predicted protein length (No. of amino acid) aresummarized in Table 1 below.

TABLE 1 AtTLP family members cDNA Predicted AGI Gene GenBank ProteinGene Name BAG Locus TC group Code Accession No. Length AtTLP1 F22K20.1TC95487 At1g76900 AF487267 455 AtTLP2 T30D6.21 TC86308 At2g18280AY045773 394 AtTLP3 F17A22.29 TC86633 At2g47900 AY045774 406 AtTLP4F8K4.13 — At1g61940 — 265 AtTLP5 T10P12.9 TC102456 At1g43640 AY046921429 AtTLP6 F8G22.1 TC90700 At1g47270 AF487268 388 AtTLP7 F12M16.22TC88599 At1g53320 AY092403 379 AtTLP8 T24D18.17 — At1g16070 AF487269 397AtTLP9 F24P17.15 TC102624 At3g06380 AF487270 380 AtTLP10 F4F7.13.TC101291 At1g25280 AF487271 445 AtTLP11 T1A4.60 — At5g18680 AY046922 380

Gene-specific 5′ and 3′ primers were designed based on the sequence ofthe predicted open reading frame (ORF) and the corresponding EST in thedatabase. The primer pairs used are listed below:

AtTLP1-5′ (5′-ATGTCGTTCCGTAGCATAGTTCGT-3′) (SEQ ID NO:23) AtTLP1-3′(5′-TTATTCGCAAGCAAGTTTTGTGTCG-3′) (SEQ ID NO:24) AtTLP2-5′(5′-ATGTCTTTGAAAAGCATCCTTCGTGATC-3′) (SEQ ID NO:25) AtTLP2-3′(5′-TTACCCTTCACATGCCGGTTTGGTGTCA-3′) (SEQ ID NO:26) AtTLP3-5′(5′-ATGTCCTTCAAGAGTCTCATTCAG-3′) (SEQ ID NO:27) AtTLP3-3′(5′-TCATTCACATGCTATCTTGGTGTC-3′) (SEQ ID NO:28) AtTLP5-5′(5′-ATGTCGTTTCTGAGTATTGTTCG-3′) (SEQ ID NO:29) AtTLP5-3′(5′-TTATTCACATGCCAATTTAGTAT-3′) (SEQ ID NO:30) AtTLP6-5′(5′-ATGTCATTGAAGAACATAGTGAA-3′) (SEQ ID NO:31) AtTLP6-3′(5′-TCATTCGCAGACTGGCTTCGTGT-3′) (SEQ ID NO:32) AtTLP7-5′(5′-ATGCCTTTGTCACGGTCCCTC-3′) (SEQ ID NO:33) AtTLP7-3′(5′-TCACTCGCAGGCAAGTTTAGTG-3′) (SEQ ID NO:34) AtTLP8-5′(5′-ATGGCTGGTTCGAGAAAAGTGAA-3′) (SEQ ID NO:35) AtTLP8-3′(5′-TCAAACAGTACAACAAAGCTTGG-3′) (SEQ ID NO:36) AtTLP9-5′(5′-ATGACGTTCCGAAGTTTACTCCA-3′) (SEQ ID NO:37) AtTLP9-3′(5′-TTATTCACAGGCAATTCTGGTTT-3′) (SEQ ID NO:38) AtTLP10-5′(5′-ATGTCGTTTCGAGGCATTGTTCA-3′) (SEQ ID NO:39) AtTLP10-3′(5′-CTATTCACAAGCAAGCTTGGTGT-3′) (SEQ ID NO:40) AtTLP11-5′(5′-ATGTCGTTTCTGAGTATTGTTCG-3′) (SEQ ID NO:41) AtTLP11-3′(5′-TTATTCACATGCCAATTTAGTAT-3′) (SEQ ID NO:42)

RT-PCR was then performed using total RNA from-2-week-old Arabidopsisseedlings. The total RNA was isolated using the TRIZOL reagent(Invitrogen) according the manufacture's direction. PolyA⁺-mRNA wasisolated using oligo (dT)-coated magnetic beads and the PolyATractsystem (Promega, Madison, Wis.). First strand cDNA was synthesized from0.5 μg PolyA⁺-mRNA using SuperScript II RNase H Reverse Transcriptase(Invitrogen) according to the protocol of the supplier.

The above-described gene-specific primer pairs were used for amplifyingcDNA of each AtTLP gene from first-strand cDNA. PCR conditions were asfollows: 3 min at 94° C.; 25 cycles of 1 min denaturation at 94° C./1min annealing at 55° C./1 min 30 s extension at 72° C. The PCR productswere purified using the QIAquick PCR purification kit (Qiagen) andsubcloned into a T-easy vector (Promega). Each of these clones wasverified by sequencing. Ten AtTLP cDNAs, AtTLPs 1-3 and AtTLPs 5-11,were successfully amplified.

It was found that, except for AtTLPs 2 and 11, the amino acid sequencesdeduced from the cDNA sequences of AtTLPs 1, 3, and 5-10 are identicalto the predicted ORFs in the database. The analysis of the AtTLP2 cDNAsequence indicated that its intron3 was located between 708-781 bpwhereas the predicted splicing sites for this intron located were 663and 766 bp. The analysis of AtTLP11 cDNA sequence showed that intron2and intron4 were located at 669-803 bp and 1334-1575 bp, respectively,whereas the computer predicted intron2 was at 621-803 bp and there wasno predicted intron4. All cDNA sequences obtained from this study weresubmitted to GenBank.

Sequence Analysis of AtTLP Proteins

The search for all known motifs in the deduced AtTLP amino acidsequences was conducted by MOTIF SCANNING (Pagni et al., 2001, NucleicAcids Res 29: 148-151). Multiple sequence alignment was performed usingClustalW (Thomopson et al., 1994). This analysis reveled that each AtTLPgene, except AtTLPs 4 and 8, had a well-conserved tubby domain at itsC-terminus. Unlike animal TLPs, which have highly diverse N-terminalsequences, each AtTLP, except AtTLP8, had a conserved F-box (51-57residues) containing domain (Pfam PF00646).

Pair-wise comparisons among the AtTLP proteins revealed that their tubbydomains shared 30% to 80% similarities. Further analyzing the tubbydomain revealed two PROSITE signature patterns: TUB1 (Prosite AccessionNo. PS01200) and TUB2 motif (Prosite Accession No. PS01201). The TUB1and TUB2 motifs were located at the C-terminal of each AtTLP protein andcontain 14 and 16 amino acid residues, respectively. These two TUBmotifs are highly conserved among TLPs from various organisms. ThoughAtTLP4 and 8 do not have obvious TUB1 and TUB2 motifs, their C-terminaltubby domains are recognizable by MOTIF SCANNING (N-score >15) (Pagni etal., 1993, Nucleic Acids Res 29: 148-151).

An obvious feature of AtTLPs is the tubby domain. In the tubby domain ofa mouse TUBBY protein, three positively-charged amino acid residues,R332, R363 and K330, were thought to be crucial for PI (4,5) P₂ binding(Santagata et al., 2001, Science 292: 2041-2050). A sequence alignmentof AtTLP tubby domains with the mouse TUBBY domain revealed a putativePI (4,5) P₂ binding domain in each AtTLP, except AtTLPs 4 and 8. Thissuggests that AtTLPs 1-3, 4-7, and 8-11 may bind to PI (4,5) P₂. It isknown that the mouse TUBBY protein is a bipartite transcriptionregulator. Its tubby domain possesses double-stranded DNA bindingactivity, and its N-terminal segment seems to modulate transcription(Boggon et al., 1999, Science 286: 2119-2125). In plants, the N-terminalregion of TLPs is quite different from that in mammal TLPs asAtTLP9-GAL4 DNA binding domain fusion protein failed to activatetranscription from a GAL4 promoter in a heterologous system.

Location and Gene Structure Comparison of the AtTLP Gene Family

Chromosome localizations of each AtTLP genes were determined using MapView available at the website of arabidopsis.org/servlets/mapper (Hualaet al., 2001, Nucleic Acids Res 29: 102-105). It was found that thegenes were not evenly distributed on chromosomes I, II, III, and V.Seven genes (AtTLPs 1, 4, 5, 6, 7, 8, and 10) were located on chromosomeI, and two genes (AtTLPs 2 and 3) were located on chromosomes II. Theother two, AtTLPs 9 and 11, were located on chromosomes II, IIIrespectively. Although most of the AtTLP genes were located onchromosome I, no local tandem repeats or gene clusters were identified.

By comparing the sequences of the RT-PCR products and the Arabidopsisgenome, the corrected exon-intron organizations of the AtTLP genes(except for AtTLP4) were determined. It was found that exon 1 containedthe sequences encoding each protein's N-terminal leading sequence, theF-box, and a nine-residue spacer between the F-box and tubby domain.This result indicated that the genes might have arisen from the sameancestral gene. The sequence encoding the C-terminal tubby domain wasfound to distribute in exons interrupted by 2 or 3 introns. On the basisof the exon and intron composition, the AtTLP genes were classified intothree groups. Each gene of the first group (AtTLPs 1, 2, 5, 6, 7, and10) contains three introns. Each of the second group, AtTLPs 3, 9 and11, contains an additional intron in the region encoding the C-terminalpart of the tubby domain. The third and the most distinct group (AtTLPs4 and 8) contain 5 and 8 introns, respectively.

Expression of AtTLP Genes

A coupled RT-PCR based assay was conducted to determine the expressionpattern of AtTLP genes. Total RNA was isolated from roots, main andlateral stems, rosette leaves, flower clusters, and green siliques of42-days-old soil-grown Arabidopsis. For each gene, a pair ofgene-specific primers was chosen, and PCR amplifications were carriedout using 15 ng of first strand cDNA synthesized as described above.Primers of ubiquitin gene, UBQ10, (5′-ATTTCTCAAAATCTTAAAAACTT-3′ (SEO IDNO:43) and 5′-TGATAGTTTTCC CAGTCAAC-3′ (SEQ ID NO:44)) were used toamplify ubiquitin, which served as an internal loading standard (Norriset al., 1993, Plant Mol. Biol. 21: 895-906).

The results showed that AtTLPs 1, 2, 3, 6, 7, 9, 10 and 11 wereexpressed in all organs tested, with slight variations in mRNAaccumulation. In contrast, AtTLPs 5 and 8 were primarily expressed inthe root, flower, and silique. The organ-specific expressions of AtTLPs5 and 8 indicate their specific roles in particular organs.

Although the expression of AtTLP1, 2, 3, 6, 7, 9, 10 and 11 is presentin all tissues tested, the possibility that these genes are expressedwith cell type specificity could not be excluded. It is possible thatdifferential expression of these AtTLP genes could only be observed wheninternal developmental programming was altered or specific environmentalstimuli were applied to the plants. To test this hypothesis, the publicArabidopsis Functional Genomics Consortium (AFGC) microarray expressiondatabase (the Stanford Microarray Database, available at the website ofstanford.edu/MicroArray/SMD/) (Wu et al., 2001, Plant Physiol Biochem39: 917-926) was searched. Twofold expression was used as the differencecutoff. Based on the search, the expression profiles of DNA fragmentscorresponding to AtTLP2, 7, 9 and 10 were summarized in Table 2 below.

TABLE 3 Microarray analysis of AtTLP genes expression Channel 1 Channel2 Ch2/Ch1 Normalized (Mean)^(b,c) Experiment Description DescriptionAtTLP2 AtTLP7 AtTLP9 AtTLP10 Hormone Effect Auxin Response msgseedlings, msg seedlings, 10 uM 0.32 2.21 untreated IAA for 30 min AuxinInduction Mock-treated NAA-treated 0.46 2.22 Columbia roots Columbiaroots Cytokinin response Control 15 min cytokinin 2.19 treatmentAbscisic acid Wild type control Abscisic acid 0.49 2.6 0.35 Insensitive1 insensitive 1 mutant edrl Mutant Wild type leaves edrl mutant leaves2.33 Downstream genes of KN1 Control Overexpression of 0.43 2.78 4.73KN1-GR in Columbia-0 background Stress Effects of Elevated Columbialeaves Columbia leaves 0.33 atmospheric CO₂ 360 ppm CO₂ 1000 ppm CO₂Genes involved in chilling Cold treated Columbia Cold treated cls8 0.220.15 tolerance wild type tissue mutant tissue Genes involved in [K+] =120 uM [K+] = 2 mM 0.34 0.2 potassium nutrition Cadmium Control 10 uMcadmium treated 2.72 plant Light Signaling Circadian rhythm time = 12.0Time = 0 hrs Time = 12 hrs 0.37 Phototropic stimulation Seedlings grownin the nph4–2 seedlings grown 2.08 dark and exposed to in the dark andexposed 1 hr blue light to 1 hr blue light Protein import into Wild typecia-2 (mutant) 3.46 chloroplasts: CIA-2 Identification of genes in WTleaves after cch1 leaves after 2.02 chlorophyll starvation exposure to230 uE exposure to 230 uE for 2 days for 2 days Stress Effects ofElevated Columbia leaves Columbia leaves 0.33 Atmospheric CO₂ 360 ppmCO₂ 1000 ppm CO₂ Genes involved Cold treated Columbia Cold treated cls80.22 0.15 In chilling tolerance wild type tissue mutant tissue Genesinvolved in [K+] = 120 uM [K+] = 2 mM 0.34 0.2 potassium nutritionCadmium Control 10 uM cadmium 2.72 treated plant ^(a)These data areobtained from the website of afgc.stanford.edu/afgc_html/site2.htm^(b)All data are corresponding with fluorescence intensities greaterthan 500 in both channels and ch2/ch1 normalized ratio ≧2.0 or ≦0.5^(c)When searching dbEST with blastn, we find Arabidopsis ESTcorresponding to fragments of four AtTLPs represented on microarray datagenerated by AFGC. AtTLP2 is corresponding to the EST clone 289B10T7 and173K22T7. AtTLP7 AtTLP9 and AtTLP10 are corresponding to the EST clone173G1T7, 201E19T7 and F3E6T7, respectively.

The resulted show that factors like hormone fluctuation andenvironmental stimuli modulate the expression of the four AtTLP genes.As shown in Table 2, the four AtTLP genes had different responses totreatments of various hormones. AtTLP2 gene expression instantaneouslyincreased more than twofold with cytokinin treatment but decreased toone-third after being treated with IAA. This suggests that Cytokinin andauxin play antagonistic roles in regulating AtTLP2 gene expression.

Another cytokinin-related experiment was aimed at identifying downstreamgenes of KN1. KN1-like protein is a homeobox transcription factor. Itsoverexpression upregulates cytokinin production and leads to delayedsenescence (Vollbrecht et al., 1991, Nature 350: 241-243). Theexpression of AtTLP7 and 10 is upregulated in KN1 overexpressiontransgenic plant while AtTLP2 is down-regulated.

The different responses of AtTLPs 7, 2, and 10 to ABA treatment is alsoworth noticing. In abscisic acid insensitive 1 mutant (Pei et al., 1997,Plant Cell 9: 409-423), the expression of AtTLP2 and AtTLP10 decreasesby two to threefold, but AtTLP7 expression increases over twofold.Interestingly, AtTLP2 and AtTLP7 behaved oppositely to auxin treatmentand in abscisic acid insensitive 1 mutant and KN1 overexpressiontransgenic plant. These two AtTLPs therefore may functionantagonistically in regulating phytohormone-signaling pathways.

The expression level of AtTLP2 rose in the edr1 (enhanced diseaseresistance 1) mutant leaves. The EDR1 gene encodes a putative MAP kinasesimilar to CTR1, a negative regulator of ethylene response inArabidopsis (Frye et al., 2001, Proc. Nat. Acad. Sci. 98: 373-378). Theedr1 mutation of Arabidopsis also confers resistance to powdery mildewdisease (Frye and Innes, 1998, Plant Cell 10: 947-956). Thus, theregulation of AtTLP2 gene expression may be associated with SA-inducibleand ethylene defense mechanism.

Environmental stresses also impose influences on the expression of AtTLPgenes. For example, similar to the cold treatment on cls8 mutant,elevated CO₂ level inhibited the expression of AtTLP2. K⁺ deficiencyaugmented the expression of AtTLPs 7 and 10 by threefold and fivefold,respectively. Heavy metal cadmium treatment stimulated the expression ofAtTLP9.

In conclusion, the expression data of these four AtTLP genes indicatetheir involvement in phytohormone and environmental stress signaling.

AtTLP9 Interacts with ASK1 Protein

Homology searches in the public databases reveal that TLPs were alsopresent in multiple plant species, including Lemna paucicostata, Oryzasativa, Cicer arietinum, maize, and Arabidopsis. Unlike animal TLPshaving highly diverse N-terminal sequences, plant TLPs had conservedF-box-containing domain. Sequence alignment of the F-box cores fromAtTLP, TIR, UFO, COI1 and the human F-box protein Skp2 revealedconserved islands separated by regions with weak homology. Many of theconserved residues correspond with those known to be important for Skpassociation (Schulman et al., 2000, Nature 408: 381-386 and Zheng etal., 2002, Nature 416: 703-709).

The F-box domain, first found in cyclin-F, interacts with the proteinSKP1, which interacts with the Cdc53 (Cullin) proteins, to form aso-called SCF complex. The F-box is involved in recruiting specificproteins (e.g., transcription activators or repressors) and targetingthem for ubiquitin-mediated proteolysis by 26S proteosome. Analysis ofthe Arabidopsis genome revealed that Arabidopsis had 21 Skp1-like, orASK, protein, which exhibited different expression patterns. Among them,ASK1 is involved in vegetative growth and reproductive development (Zhaoet al., 2003, Plant Physiology 133: 203-217).

To test whether AtTLP could interact with ASK1, AtTLP9 was examined bythe yeast two-hybrid analysis. Yeast two-hybrid vectors, pAD-GAL4-2.1and pBD-GAL4 Cam (Stratagene, La Jolla, Calif.), were used forC-terminal GAL4 AD and BD fusion constructions, respectively. A 1.1-kbSalI-PstI fragment containing the entire coding region of AtTLP9 wascloned into the SalI-PstI site of the pBD-GAL4 Cam vector. A 480-bpEcoRI-PstI fragment containing the entire coding region of ASK1(Atg175950) was cloned into the EcoRI-PstI site of the pAD-GAL4-2.1vector. The yeast strain YRG-2 [MATa ura3-52 his3-200 ade2-101 lys2-801trp1-901 leu2-3,112 gal4-542 gal80-538 LYS2::GAL1UAS-GAL1TATA-HIS3URA3::(GAL43×17mer)-CYC1TATA-lacZ] was co-transformed with the twovectors. The Y2H analysis was performed according to the manufacturer'srecommendations (Stratagene). The result suggested that AtTLP9physically interacts with ASK1 to form SCF complex and acts as a factorfor substrate recognition in the ubiquitin-mediated proteolysis.

Attlp9 Null Mutants and Overexpression Lines

ATLP9 was analyzed to investigate for it in vivo functions. Bothloss-of-function and overexpression approaches were taken to address itsbiological roles.

To identify attlp9 T-DNA insertion mutant, AtTLP9 (At3g06380) was usedto search the T-DNA Express database at the website ofsignal.salk.edu/cgi-bin/tdnaexpress. Two attlp9 T-DNA insertion mutants(ABRC seed stock SALK_(—)016678 and 051138) were identified anddesignated as attlp9-1 and attlp9-2. T3 seeds of attlp9-1 and attlp9-2were obtained from the Arabidopsis Biological Resource Center (OhioState University, Columbus). The position of the T-DNA within the AtTLP9gene was re-confirmed by sequencing a PCR-amplified fragment amplifiedby primer pairs corresponding to the T-DNA left borders and the AtTLP9gene specific primer. The following primer pairs were used for attlp9-1and attlp9-2 specific amplification,

(SEQ ID NO:45) attlp9-1: N1, 5′-ATGACGTTCCGAAGTTTACTC-3′; (SEQ ID NO:46)LBa1, 5′-TGGTTCACGTAGTGGGCCATC-3′; (SEQ ID NO:47) attlp9-2: C1,5′-TTATTCACAGGCAATTCTGGT-3′; and (SEQ ID NO:46) LBa1,5′-TGGTTCACGTAGTGGGCCATC-3′.

It was found that Attlp9-1 had a T-DNA insertion in the coding sequenceat codon 705, whereas attlp9-2 had an insertion in the 5′ distal regionof this gene. The T-DNA insertion site of attlp9-1 was identical to thatoriginally described in the T-DNA Express database. However, the T-DNAinsertion site of attlp9-2 was in the promoter region instead of exon1as predicted in the database (the latter is supported by a potentialfull length cDNA corresponding to At3g06380 generated in RIKEN,accession number BT004092).

Southern blot was conducted with the nptII marker gene to determine theT-DNA insertion number in attlp9-1 and 9-2 knockout mutants. It wasfound that one and three T-DNA insertion events in the T₄ attlp9-1 andattlp9-2 T-DNA insertion mutants, respectively.

The T-DNAs in attlp9-1 and attlp9-2 carried a gene leading to resistanceto kanamycin. Homozygous analyses of attlp9-1 and attlp9-2 plants werecarried out by kanamycin selection and PCR based method. RT-PCR analysesof T₄ homozygous of attlp9-1 and attlp9-2 plants indicated that attlp9-1was a null allele, whereas attlp9-2 was somewhat leaky. For thephenotype investigation, attlp9-1 and attlp9-2 T₄ homozygous lines wereused for detailed analysis.

Transgenic plants with overexpressing AtTLP9 were generated. AnXbaI-SmaI fragment of AtTLP9 was inserted into an XbaI-SmaI site of thepBI121 Ti-vector (Clontech) to generate a 35S::AtTLP9 sense construct.The XbaI-SmaI fragment contained the entire AtTLP9 coding region and wasunder the control of the 35S promoter of cauliflower mosaic virus. Theconstructs were introduced into Agrobacterium strain LBA4404 byelectroporation and transformed into wild-type plants by the floral dipmethod (Clough et al., 1998, Plant J 16: 735-743). 38 independenttransgenic lines (T₀ generation) were obtained. Among them, sevenindependent homozygous lines from the T₃ sense transgenic plants wereanalyzed for the AtTLP9 expression. Each of these lines contained asingle copy of the transgene. Two independent transgenic lines (S13-2and S16-1) showed dramatic increases in the AtTLP9 transcript levels andwere further analyzed. A number of control transgenic lines weregenerated by transforming with Agrobacterium with PBI121 vector alone.

The wild type Arabidopsis thaliana ecotype Columbia-0 (Col-0) and mutantabi4-1 (obtained from Dr Wan-Hsing Cheng, Institute of Botany, AcademiaSinica Taipei) were used. The phenotypes of abi4-1 were confirmed asdescribed (Söderman et al., 2000, Plant Physiol. 124: 1752-1765) priorto use.

All seeds of the above-described lines were surface sterilized with 70%ethanol for 30 s and then with 6% household bleach for 5 min beforebeing washed five times with sterile water. For aseptic growth, theywere plated on solid medium containing Murashige and Skoog salts(Invitrogen), vitamins (Duchefa), 0.7% phytoagar (Invitrogen), and 1%sucrose and transferred to a tissue culture room. For soil growth,seedlings were transferred into individual pots 8-10 days aftergermination and maintained in the growth chamber. Plants were grown at22° C. under a 16-hr-light/8-hr-dark photoperiod aseptically or on soil.

The general development and growth phenotypes of the attlp9-1 andattlp9-2 knockout plants appear to be similar to those of the wild typeplants. However, when seeds were plated on nutrient agar media, thegermination time of mutant attlp9-1 and attlp9-2 seeds was advancedseveral hours compared with that of the wild type plants, whereas theselected sense line seeds (i.e., S13-2 and S16-1) germinated later thanvector control seeds. It was found that 50% of the wild type seedsgeminated after about 37 hour after plating. In contrast, 50% of theattlp9-1 and attlp9-2 knockout seeds geminated at hours 26-28 afterplating, and 50% of the S13-2 and S16-1 seeds geminated around hours40-42 hours after plating.

Effect of ABA on Seed Germination of Attlp9 Mutants and OverexpressionLines

It is known that seed germination is the outcome of an integration ofmany signals coordinated by the interactions of stage-specificdevelopmental regulators and the competing effects of hormonal signals(Finkelstein et al., 2002, Curr. Opin. Plant. Biol. 5: 26-32). The mostcritical hormone promoting embryo maturation and preventing germinationis ABA.

To determine whether the transgenic plants display altered ABAresponses, the above-described lines were germinated on media containingvarious concentrations of ABA. Seeds collected at the same or similartimes were used. After surface-sterilization, sterile seeds weresuspended in 0.15% agarose, and kept in the dark at 4° C. for 3 days tobreak residual dormancy. The seeds were then plated on agar plates insix replicates containing no ABA or 0.25, 0.5, 0.75, or 1.0 μM ABA in12-cm plastic petri dishes. Each agar plate was divided into sevensections, and 50 seeds of WT and AtTLP9 transgenic seeds were plated oneach section. A seed was regarded as germinated when the radicleprotruded through the seed coat.

In the presence of 1 μM ABA, the germination of sense lines seeds wasfurther delayed and the germination rate was reduced to less than 10%.In contrast, the germination rate of attlp9-1 and attlp9-2 mutant seedsnearly reached 50%, and about 30% of wild-type seeds were able togerminate in the presence of 1 μM ABA. These results suggest that thedisruption of the AtTLP9 gene affects the sensitivity of seeds toexogenous ABA.

In addition to reducing seed germination rate, ABA also inhibited thegrowth and the greening process in cotyledons of the sense transgeniclines. In MS agar medium containing 1 μM ABA and 1% sucrose, 90% of the10-d-old seedlings showed developmental arrest although the radicles ofmost sense lines seeds emerged. In contrast, under the same conditions,attlp9-1 and attlp9-2 plants continued to grow and about 45% of theseedlings continued to develop true leaves, although at slower ratesthan abi4-1 mutant does. These results indicate that the alteration ofAtTLP9 modulate plant's sensitivity to ABA during seed germination andearly seedling development.

AtTLP9 Expression is Transiently Up-regulated During Imbibition of Seeds

Real-time PCR experiments were conducted to quantify AtTLP9 transcriptlevels at seed maturation, seed germination, and early developmentstage. UBQ10 was used as the endogenous control (Norris et al., 1993,Plant. Mol. Biol. 21: 895-906). Primers were designed using PrimerExpress 1.0 software (Applied Biosystems). The primers used were:

AtTLP9 forward primer, (SEQ ID NO:48) 5′-TAGGCCACACCGTGTAGTTCA-3′;AtTLP9 reverse primer, (SEQ ID NO:49) 5′-CGTCAACAGTCTCAACCCTAATCA-3′;UBQ10 forward primer, (SEQ ID NO:50) 5′-AGAAGTTCAATGTTTCGTTTCATGTAA-3′;and UBQ10 reverse primer, (SEO ID NO:51)5′-GAACGGAAACATAGTAGAACACTTATTCA-3′.

The real-time PCR was performed in a 50 μL reaction mixture containing500 ng first strand cDNA, 2.5 μM each primers and 1× SYBR Green PCRMaster Mix (Applied Biosystems). PCR cycling was 50° C. for 2 min, 95°C. for 10 min, followed by 40 cycles of 15 sec at 95° C./1 min at 60° C.The UBQ10 mRNA quantity was set at ‘1’ and AtTLP9 expression wasdetermined relative to control samples. Threshold cycles were determinedby Sequence Detection System V. 1.7a software (Applied Biosystems). Theproducts were quantified by the ABI PRISM 7700 Sequence Detection System(Applied Biosystems, Scoresby, Victoria, Australia).

Seed germination is divided into three phases: imbibition, increasedmetabolic activity, and initiation of growth (Bewley. 1997, Plant Cell9: 1055-1066). It was found that during seed maturation and seedimbibition at 4° C. for 72 h, AtTLP9 transcripts remained at arelatively low level. When the seeds were transferred to 22° C. forfurther incubation, the levels rose after 8 h, peaked at 16 h, and fellrapidly after 24 h when the radicle emerged. The AtTLP9 transcripts werebarely detectable afterwards. The transient expression of AtTLP9indicated that AtTLP9 functions at stage II of seed germination as acheckpoint before radicle protrusion.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination.

Each feature disclosed in this specification may be replaced by analternative feature serving the same, equivalent, or similar purpose.Thus, unless expressly stated otherwise, each feature disclosed is onlyan example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the scope of thefollowing claims.

1. A transformed plant cell comprising a recombinant nucleic acid thatencodes the heterologous polypeptide of SEQ ID NO:
 9. 2. A transgenicplant comprising a recombinant nucleic acid that encodes theheterologous polypeptide of SEQ ID NO:
 9. 3. A method of producing atransformed plant cell, the method comprising: introducing into a plantcell a recombinant nucleic acid encoding the heterologous polypeptide ofSEQ ID NO: 9, and expressing the polypeptide in the cell.
 4. A method ofproducing a transgenic plant, the method comprising: introducing into aplant cell a recombinant nucleic acid encoding the heterologouspolypeptide of SEQ ID NO:9, expressing the polypeptide in the cell, andcultivating the cell to regenerate a plant.
 5. A transformed plant cellcomprising a heterologous DNA sequence that encodes a polypeptide havingat least 95% sequence identity to SEQ ID NO:9, and wherein expression ofthe DNA sequence in said plant cell confers increased sensitivity to ABArelative to an untransformed plant cell of the same species.
 6. A methodof producing a transformed plant cell, the method comprising introducinginto a plant cell a heterologous DNA sequence that encodes a polypeptidehaving at least 95% sequence identity to SEQ ID NO:9, and whereinexpression of the DNA sequence in said plant cell confers increasedsensitivity to ABA relative to an untransformed plant cell of the samespecies.
 7. A method of producing a transgenic plant, the methodcomprising introducing into a plant cell a heterologous DNA sequencethat encodes a polypeptide having at least 95% sequence identity to SEQID NO:9, and cultivating the transformed cell to regenerate thetransgenic plant, wherein expression of the DNA sequence in said plantconfers increased sensitivity to ABA relative to an untransformed plantof the same species.